Defective performance of the endosomal/endocytic system is associated with numerous diseases, including diabetes, cancer and infection, yet the molecular mechanisms that govern proper regulation of endosome membrane dynamics are elusive. Several known molecular components of the mammalian cell endocytic machinery appear to be proteins that bind or synthesize phosphoinositides. PIKfyve, a product of an evolutionarily conserved single-copy gene, identified by our group, has both these features. It binds to membrane phosphatidylinositol (PI)3P and synthesizes PI3,5P2. PIKfyve mutants defective in PI3,5P2 synthesis or PIKfyve protein ablation in mammalian cells induce enormous cytoplasmic vacuoles of endocytic origin, suggesting enzymes's vital functions. This is underscored by the embryonic lethality of C. elegans and Drosophila PIKfyve null mutants. The detailed molecular/cellular mechanism whereby PIKfyve and its product PI3,5P2 control the proper dynamics of endosomal/endocytic membranes is, however, unknown. The long- term goal of our research is to reveal the pleiotropic functions of both the PIKfyve protein and the products of its enzymatic activity, as well as the physiological, molecular and cellular bases for those functions. The objective of this proposal is to determine how PIKfyve and its newly identified protein partners govern proper endosome dynamics and functioning of the endosomal/endocytic transport systems. Our central hypothesis is that PIKfyve-catalyzed production of PI3,5P2, regulated by the physically associated PIKfyve protein partners, ArPIKfyve and Sac3, promotes dynamic early endosome remodeling to trigger formation of carrier vesicles that then move on microtubules to appropriate destinations in a process dependent on PIKfyve's physical interaction with a motor protein adapter JIP4. Our central hypothesis has been formulated on the basis of strong preliminary unpublished data generated by our group during the previous funding cycle. We will test the central hypothesis by pursuing four specific aims: 1) identify a PIKfyve-ArPIKfyve-Sac3 physical interaction and role of the Sac3 phosphatase in PI3,5P2 turnover;2) identify a key role for the PIKfyve/Sac3-controlled PI3,5P2 homeostasis in early endosome membrane plasticity;3) identify PIKfyve's physical association with the JIP4L kinesin adapter and role in microtubule-based endosome vesicle motility;4) determine how changes in PIKfyve, ArPIKfyve and/or Sac3 protein levels affect insulin-regulated glucose transporters'membrane dynamics and glucose transport in adipocyte models. These aims will be addressed by exploiting the most appropriate and sensitive currently available techniques for modulating intracellular protein levels or functions, analyzing cellular phosphoinositide contents, inspecting the morphology of the endocytic membrane compartments and monitoring the intracellular vesicle motility in real time. The proposed work is innovative because it focuses on the evolutionally conserved PIKfyve pathway, first identified by our group in mammalian cells, and will provide a novel conceptual framework for its role in endosome membrane dynamics.